Steroid-binding Activity Research Articles

Neutrophil elastase-cleaved corticosteroid-binding globulin is absent in human plasma.

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.

Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90β in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90β, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90β in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90β have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90αα and GR•Hsp90ββ homodimers and no GR•Hsp90αβ heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90αβ heterodimers. The formation of GR•Hsp90αβ heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αβ heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.

Functional implications of corticosteroid-binding globulin N-glycosylation.

Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six N-glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are N-glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved N-glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one N-glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its N-glycan is removed. When expressed in N-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2–4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an N-glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the Pseudomonas aeruginosa protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, N-glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.

Corticosteroid-binding globulin is a biomarker of inflammation onset and severity in female rats.

Plasma corticosteroid-binding globulin (CBG) plays a critical role in regulating glucocorticoid bioavailability and is an acute phase 'negative' protein during inflammation. In an adjuvant-induced arthritis model, plasma CBG levels decrease in rats that develop severe inflammation, and we have now determined when and how these reductions in CBG occur. After administering complete Freund's adjuvant or saline intra-dermally at the tail base, blood samples were taken periodically for 16days. In adjuvant-treated rats, decreases in plasma CBG levels matched the severity of inflammation, and decreases were observed 4days before any clinical signs of inflammation. Decreases in CBG levels coincided with an ~5kDa reduction in its apparent size, consistent with proteolytic cleavage, and cleaved CBG lacked steroid-binding activity. At the termination of the experimental period, hepatic Cbg mRNA levels were decreased in rats with severe inflammation. While plasma TNF-α increased in all adjuvant-treated rats, increases in Il-4, IL-6, IL-10, IL-13 and IFN-γ were only observed in rats with cleaved CBG. Rats with cleaved CBG also exhibited increased spleen weights, and strong negative correlations were observed among CBG, IL-6 and spleen weights, respectively. However, there were no differences in hepatic Cbg mRNA levels in relation to the apparent proteolysis of CBG, suggesting that CBG cleavage occurs before changes in hepatic Cbg expression. Our results indicate that the levels and integrity of plasma CBG are biomarkers of the onset and severity of inflammation. Dynamic changes in the levels and function of CBG likely modulate the tissue availability of corticosterone during inflammation.

Nucleotide Dependency of Endogenous Hsp90 Chaperone Protein Machinery and Its Association with Baculovirally Expressed Glucocorticoid Receptor

Hsp90 and hsp70 are essential eukaryotic chaperone proteins required for facilitating functional activity of mammalian transcription factors, including the glucocorticoid receptor (GR). Both chaperones exist in ATP‐ and ADP‐bound states, and understanding the relationship between nucleotide‐bound forms of hsp90 and hsp70, their affiliated cochaperones, and GR activation has significant pharmacological implications. The goal of this study was to investigate the extent to which hsp90 and hsp70 nucleotide interactions affect production of a GR‐hsp90 heterocomplex capable of steroid binding activity. Reticulocyte lysate (RL) containing high endogenous concentrations of hsp90 and hsp70 was incubated in the presence or absence of an ATP regenerating system and fractionated by size exclusion chromatography (SEC). ATP incubation did not appear to alter total global protein elution, as detected by A280 spectrophotometry. However, SDS‐PAGE and western blot analysis identified nucleotide‐associated alterations in hsp90 SEC elution profiles. In order to further compare nucleotide dependency and chaperone effects, full‐length GR, produced in baculovirally infected insect cells, was immunoadsorbed, stripped of endogenous chaperones, and reassembled into a heterocomplex with hsp90 ± ATP. SDS‐PAGE and western blotting showed hsp90 derived from RL−ATP samples were tightly bound to the GR, while hsp90 from RL+ATP failed to stay bound to the GR. The difference in SEC elution profiles and co‐immunoadsorption imply a notable role of nucleotide alterations in the hsp90 chaperone machinery and functional interactions with the GR.

Hsp90 chaperone machinery coexpression increases functional glucocorticoid receptor protein expression, receptor•hsp90 heterocomplex formation, and steroid binding activity

While hsp90 client proteins such as the glucocorticoid receptor (GR) are highly expressed using a baculovirus expression system, the majority of overexpressed protein is misfolded and nonfunctional. Nonaggregated‐GR forms a functional GR•hsp90 heterocomplex with steroid binding activity. We hypothesized baculoviral co‐overexpression of the complete hsp90 chaperone machinery (i.e. hsp90, hsp70, Hop, hsp40, and p23) would decrease overexpressed client protein aggregation and increase functional client protein concentration and activity. A fusion protein containing wild‐type GR ligand binding domain (GST‐LBD) was produced using a baculovirus expression system in the presence or absence of coinfected baculovirus expressing the hsp90 chaperone machinery. Western blot and steroid binding analysis demonstrated baculoviral coexpression of hsp90 chaperone machinery elicited a >;2‐fold increase in client protein expression and a >;7.5‐fold increase in steroid binding activity. Coexpression of the hsp90 chaperone machinery increased formation of functional GST‐LBD•hsp90 heterocomplexes and steroid binding capacity following in vitro reconstitution. These data show baculoviral coinfection with hsp90 chaperone machinery increases overexpressed GR LBD folding and function. We speculate this methodology may be applicable to baculoviral expression of many, if not all, hsp90 client proteins.

Single nucleotide polymorphisms in the human corticosteroid-binding globulin promoter alter transcriptional activity

Corticosteroid-binding globulin (CBG) is a high-affinity plasma protein that transports glucocorticoids and progesterone. Others and we have reported non-synonymous single nucleotide polymorphisms (SNPs) that influence CBG production or steroid-binding activity. However, no promoter polymorphisms affecting the transcription of human CBG gene (Cbg) have been reported. In the present study we investigated function implications of six promoter SNPs, including -26 C/G, -54 C/T, -144 G/C, -161 A/G, -205 C/A, and -443/-444 AG/-, five of which are located within the first 205 base pairs of 5'-flanking region and close to the highly conserved footprinted elements, TATA-box, or CCAAT-box. Luciferase reporter assays demonstrated that basal activity of the promoter carrying -54 T or -161 G was significantly enhanced. The first three polymorphisms, -26 C/G, -54 C/T, and -144 G/C located close to the putative hepatic nuclear factor (HNF) 1 binding elements, altered the transactivation effect of HNF1β. We also found a negative promoter response to dexamethasone-activated glucocorticoid receptor (GR) α, although none of the SNPs affected its transrepression function. Our results suggest that human Cbg -26 C/G, -54 C/T, -144 G/C, and -161 A/G promoter polymorphisms alter transcriptional activity, and further studies are awaited to explore their association with physiological and pathological conditions.

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways (1,2). Of these many 'client' proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid (3). Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored (4) . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [(3)H]steroid. Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 (5). A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source (6). In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K(+), which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity (7). This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding (8-11).

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Methoxychlor affects multiple hormone signaling pathways in the largemouth bass ( Micropterus salmoides) liver

Methoxychlor (MXC) is an organochlorine pesticide that has been shown to have estrogenic activity by activating estrogen receptors and inducing vitellogenin production in male fish. Previous studies report that exposure to MXC induces changes in mRNA abundance of reproductive genes in the liver and testes of largemouth bass ( Micropterus salmoides). The objective of the present study was to better characterize the mode of action of MXC by measuring the global transcriptomic response in the male largemouth liver using an oligonucleotide microarray. Microarray analysis identified highly significant changes in the expression of 37 transcripts ( p < 0.001) (20 induced and 17 decreased) in the liver after MXC injection and a total of 900 expression changes ( p < 0.05) in transcripts with high homology to known genes. Largemouth bass estrogen receptor alpha ( esr1) and androgen receptor ( ar) were among the transcripts that were increased in the liver after MXC treatment. Functional enrichment analysis identified the molecular functions of steroid binding and androgen receptor activity as well as steroid hormone receptor activity as being significantly over-represented gene ontology terms. Pathway analysis identified c-fos signaling as being putatively affected through both estrogen and androgen signaling. This study provides evidence that MXC elicits transcriptional effects through the estrogen receptor as well as androgen receptor-mediated pathways in the liver.

Functional Redundancy of Steroid C26-monooxygenase Activity in Mycobacterium tuberculosis Revealed by Biochemical and Genetic Analyses

One challenge to the development of new antitubercular drugs is the existence of multiple virulent strains that differ genetically. We and others have recently demonstrated that CYP125A1 is a steroid C(26)-monooxygenase that plays a key role in cholesterol catabolism in Mycobacterium tuberculosis CDC1551 but, unexpectedly, not in the M. tuberculosis H37Rv strain. This discrepancy suggests that the H37Rv strain possesses compensatory activities. Here, we examined the roles in cholesterol metabolism of two other cytochrome P450 enzymes, CYP124A1 and CYP142A1. In vitro analysis, including comparisons of the binding affinities and catalytic efficiencies, demonstrated that CYP142A1, but not CYP124A1, can support the growth of H37Rv cells on cholesterol in the absence of cyp125A1. All three enzymes can oxidize the sterol side chain to the carboxylic acid state by sequential oxidation to the alcohol, aldehyde, and acid. Interestingly, CYP125A1 generates oxidized sterols of the (25S)-26-hydroxy configuration, whereas the opposite 25R stereochemistry is obtained with CYP124A1 and CYP142A1. Western blot analysis indicated that CYP124A1 was not detectably expressed in either the H37Rv or CDC1551 strains, whereas CYP142A1 was found in H37Rv but not CDC1551. Genetic complementation of CDC1551 Δcyp125A1 cells with the cyp124A1 or cyp142A1 genes revealed that the latter can fully rescue the growth defect on cholesterol, whereas cells overexpressing CYP124A1 grow poorly and accumulate cholest-4-en-3-one. Our data clearly establish a functional redundancy in the essential C(26)-monooxygenase activity of M. tuberculosis and validate CYP125A1 and CYP142A1 as possible drug targets.

Novel Corticosteroid-Binding Globulin Variant That Lacks Steroid Binding Activity

Corticosteroid-binding globulin (CBG) is the principal carrier for glucocorticoids in the circulation and a regulator of their bioavailability. Inherited CBG deficiencies are rarely reported, and only three causative mutations in four families have been described. In a 26-yr-old female with hypotension, fatigue, and undetectable total serum cortisol at presentation, we have identified a novel homozygous c.776g>t transversion in exon 3 of the CBG (SERPINA6) gene. This results in a p.Gly237Val substitution that is predicted to influence the positioning of two β-sheets that constitute part of the CBG steroid-binding site. Two siblings were also homozygous for the variant, whereas her mother and an unaffected sibling were heterozygous. No other symptomatic family members were identified apart from the proband. Individuals homozygous for the variant had serum CBG levels below the reference range when measured by RIA, but CBG was unmeasurable in cortisol-binding capacity assays. In the same individuals, we observed very low baseline and stimulated total serum cortisol levels but normal free serum and salivary cortisol and plasma ACTH. In a study of ultradian cortisol pulsatility, increased pulse frequency was only observed in the proband. We describe a novel CBG variant that lacks steroid binding activity. All mutant homozygotes have very low total serum cortisol, but normal free serum cortisol levels. The only biochemical feature to distinguish the symptomatic subject was increased cortisol pulsatility, and we suggest that this may influence glucocorticoid signaling and contribute to symptoms previously associated with CBG deficiency.

Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

Testosterone-inducible Regulator Is a Kinase That Drives Steroid Sensing and Metabolism in Comamonas testosteroni

The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.

Regulation of the Dynamics of hsp90 Action on the Glucocorticoid Receptor by Acetylation/Deacetylation of the Chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 "client" protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601-607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR.hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR.hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR.hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR.hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR.hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a approximately 100-fold shift to the right in the steroid dose response for gene activation.

Expression of Rabbit Sex Hormone-Binding Globulin during Pregnancy and Prenatal Development and Identification of a Novel Isoform

SHBG is a homodimeric plasma glycoprotein. It functions as a carrier for sex steroids in blood and regulates their access to target cells. In human and rabbit, SHBG is a single-copy gene comprised of eight exons and is expressed primarily in the liver and testis. In the present study, the ontogeny of rabbit SHBG (rbSHBG) gene expression was examined in both fetus and mothers. Trace amounts of rbSHBG mRNA were detected in fetal liver from d 11 to d 29 gestation. These levels increased dramatically at d 30 and remained high until parturition (d 33). In contrast, high levels of rbSHBG mRNA were detected in the maternal liver early during pregnancy, with maximal levels being attained by d 22 and declining markedly thereafter. A rbSHBG transcript lacking the exon 4 sequences was consistently expressed along with the rbSHBG mRNA. When expressed as a glutathione-S-transferase-fusion protein, this alternatively spliced rbSHBG transcript resulted in a product with almost no steroid binding activity, unlike the full-length rbSHBG-glutathione-S-transferase fusion protein, which bound 5alpha-dihydrotestosterone. Antibody specific to the novel rbSHBG isoform lacking the exon 4-encoding domain was raised, and a single immunoreactive protein of 33-35 kDa was detected by Western blot analysis in both fetal and maternal liver, and this indicates that the rbSHBG transcripts lacking exon 4 sequences are translated in vivo. An RT-PCR analysis further revealed that this alternatively spliced SHBG transcript is present in human HepG2 cells as well as human and mouse testes, indicating that exon 4 splicing in SHBG transcription is conserved among mammalian species. To our knowledge, this is the first report of the identification of a SHBG exon 4 splice variant that is translated. Because the SHBG isoform it encodes lacks appreciable steroid-binding activity, it may function beyond that of the widely accepted role of SHBG as a steroid-transport protein.

The Role of hsp90 in Heme-dependent Activation of Apo-neuronal Nitric-oxide Synthase

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the ubiquitous protein chaperone hsp90. We have shown previously that nNOS expressed in Sf9 cells where endogenous heme levels are low is activated from the apo- to the holo-enzyme by addition of exogenous heme to the culture medium, and this activation is inhibited by radicicol, a specific inhibitor of hsp90 (Billecke, S. S., Bender, A. T., Kanelakis, K. C., Murphy, P. J. M., Lowe, E. R., Kamada, Y., Pratt, W. B., and Osawa, Y. (2002) J. Biol. Chem. 278, 15465-15468). In this work, we examine heme binding by apo-nNOS to form the active enzyme in a cell-free system. We show that cytosol from Sf9 cells facilitates heme-dependent apo-nNOS activation by promoting functional heme insertion into the enzyme. Sf9 cytosol also converts the glucocorticoid receptor (GR) to a state where the hydrophobic ligand binding cleft is open to access by steroid. Both cell-free heme activation of purified nNOS and activation of steroid binding activity of the immunopurified GR are inhibited by radicicol treatment of Sf9 cells prior to cytosol preparation, and addition of purified hsp90 to cytosol partially overcomes this inhibition. Although there is an hsp90-dependent machinery in Sf9 cytosol that facilitates heme binding by apo-nNOS, it is clearly different from the machinery that facilitates steroid binding by the GR. hsp90 regulation of apo-nNOS heme activation is very dynamic and requires higher concentrations of radicicol for its inhibition, whereas GR steroid binding is determined by assembly of stable GR.hsp90 heterocomplexes that are formed by a purified five-chaperone machinery that does not activate apo-nNOS.

The Hsp90 Cochaperone p23 Is the Limiting Component of the Multiprotein Hsp90/Hsp70-based Chaperone System in Vivo Where It Acts to Stabilize the Client Protein·Hsp90 Complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.

Photoaffinity labeling identification of thyroid hormone-regulated glucocorticoid-binding peptides in rat liver endoplasmic reticulum: an oligomeric protein with high affinity for 16β-hydroxylated stanozolol

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic actions of the17α-alkylated testosterone derivative (17α-AA) stanozolol (ST). We have previously reported that male rat liver endoplasmic reticulum contains two steroid-binding sites associated with high molecular mass oligomeric proteins: (1) the ST-binding protein (STBP); and (2) the low-affinity glucocorticoid-binding protein (LAGS). To further explore the role of LAGS on the mechanism of action of ST, we have now studied: (1) the interaction of ST and its hydroxylated metabolites with solubilized LAGS and the cytosolic glucocorticoid receptor (GR); and (2) the effects of hormones on the capability of STBP to bind ST. We found that, unlike 17α-methyltestosterone, neither ST nor its hydroxylated metabolites bind to GR. However, the 16β-hydroxylation of ST significantly increases the capability of LAGS to bind ST. Interestingly, 3′-hydroxylation of ST abrogates the capability of LAGS to bind ST. ST ( k i =30 nM) and 16β-hydroxystanozolol ( k i =13 nM) bind with high affinity to LAGS, and are capable of accelerating the rate of dissociation of previously bound dexamethasone from the LAGS. STBP and LAGS are strongly induced by ethinylestradiol. However, unlike STBP, LAGS is regulated by thyroid hormones and growth hormone, which proves that these steroid-binding activities are associated with different binding sites. These findings seem to suggest a novel mechanism for ST whereby membrane-associated glucocorticoid-binding activity is targeted by the 16β-hydroxylated metabolite of ST. ST and its 16β-hydroxylated metabolite modulate glucocorticoid activity in the liver through negative allosteric modulation of LAGS, with the result of this interaction an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent formation of a GR.hsp70 complex that primes the receptor for subsequent ATP-dependent activation by hsp90, Hop, and p23. This study focuses on three aspects of the GR priming reaction with hsp70. First, we have visualized the primed GR.hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size approximately 1:2 and a few complexes of larger size. Second, in a recent study of progesterone receptor priming, it was shown that hsp40 binds first, leading to the notion that it targets hsp70 to the receptor. We show here that hsp40 does not perform such a targeting function in priming the GR. Third, we focus on a short amino-terminal segment of the ligand binding domain that is required for GR.hsp90 heterocomplex assembly. By using two glutathione S-transferase (GST)/ligand binding domain fusions with (GST/520C) and without (GST/554C) hsp90 binding and steroid binding activity, we show that the priming step with hsp70 occurs with GST/554C, and it is the subsequent assembly step with hsp90 that is defective.